Protein O-mannosylation is evolutionarily conserved from bacteria to humans (Lommel and Strahl 2009). It is known that in fungi, the majority of secreted and cell wall proteins are highly O-mannosylated (Strahl-Bolsinger, Gentzsch et al. 1999). So far, in humans, there are total of 50 proteins harboring O-mannosyl glycan including dystroglycan (DG) (Endo 2003; Hu, Li et al. 2011), 37 of cadherins, 6 of plexins (Baenziger 2013), 4 of lecticans (aggrecan, brevican, neurocan, and versican) (Pacharra, Hanisch et al. 2013), CD24 (Bleckmann, Geyer et al. 2009), PTPRZ1 (Dwyer, Baker et al. 2012), and neurofascin 186 (Pacharra, Hanisch et al. 2012). Most of these proteins are either extracellular matrix (ECM) proteins, or the receptors of ECM proteins. The knowledge of protein O-mannosylation in mammalian cells are mainly from studies on DG that was originally isolated as a laminin binding protein named as Crainin from embryonic chicken brain cells (Smalheiser and Schwartz 1987). It is a ubiquitously expressed plasma membrane associated receptor and abundant in skeletal muscles and heart. In muscles, DG associates with dystrophin and other glycoproteins forming a large protein complex so called dystrophin-associated glycoproteins complex (DAGC) (Ervasti, Ohlendieck et al. 1990). Dystroglycan is encoded by DAG1 gene that produces one mRNA and is translated into a single polypeptide that is cleaved into two subunits: α-DG and β-DG (Ibraghimov-Beskrovnaya, Ervasti et al. 1992). The two subunits remain tightly associated in a non-covalent manner as the key components of the DAGC. α-DG is located on the cell surfaces and serves as the receptor for several ECM proteins such as agrin (Bowe, Deyst et al. 1994), laminins (Smalheiser and Schwartz 1987), and perlecan (Schneider, Khalil et al. 2006). β-DG protein contains a transmembrane domain and its cytoplasmic tail interacts with cytoskeletal proteins, such as dystrophin, plectin, dystrobrevin, and other signaling molecules (Yang, Jung et al. 1995; Rezniczek, Konieczny et al. 2007; Swiderski, Shaffer et al. 2014). Thus, through their interactions with other proteins, α-DG and β-DG connect the ECM and the cytoskeletons, which plays an important role in muscle functions (Ervasti and Campbell 1993). α-DG is involved in many physical and pathological processes such as development (Durbeej, Larsson et al. 1995), muscular dystrophies (Brockington, Yuva et al. 2001), cancer progression (Bao, Kobayashi et al. 2009), and viral infections (Barresi and Campbell 2006). Importantly, α-DG is highly glycosylated with multiple forms of glycans, one of which is O-mannosyl glycan that is essential for its ligand binding activity, hereafter referred to it as the functional O-mannosyl glycan (FOG) (Alhamidi, Kjeldsen Buvang et al. 2011). Deficiency of the FOG on α-DG also called hypoglycosylation of α-DG impairs its ligand binding activity and results in various disorders including muscular dystrophies and cancer progression for review see the reference (Wells 2013).
Muscular Dystrophies
Muscular dystrophies (MD) are a large group of genetically heterogeneous diseases with a wide spectrum of clinical manifestations, which are characterized by progressive muscle weakness and wasting. Most MD patients eventually lose their mobility and many of them die prematurely. To date, there is no effective treatment for any type of MD. The majority of MDs are caused by genetic defects in the genes involved in the connections between ECM and cytoskeletons in muscles (Xiong, Kobayashi et al. 2006). The genetic defects underlining these MDs can be divided to the following groups. 1) The defects of the ECM proteins such as collagen VIα1-3 subunits and laminin α2 chain cause Bethlem myopathy and congenital muscular dystrophy (CMD), respectively (Xu, Wu et al. 1994; Camacho Vanegas, Bertini et al. 2001). 2) The defects of dystrophin (Hoffman, Brown et al. 1987) or other components of DAGC such as α- through δ-Sarcoglycans cause DMD/BMD and various limb-girdle muscular dystrophies (LGMDs), respectively (Hoffman, Brown et al. 1987; Lim and Campbell 1998; Hara, Balci-Hayta et al. 2011). 3) The defects of the proteins involved in O-mannosylation of α-DG: such as Fukutin (Kobayashi, Nakahori et al. 1998), Fukutin relate protein (FKRP) (Esapa, Benson et al. 2002), LARGE (van Reeuwijk, Grewal et al. 2007), POMT1/2 (Beltran-Valero de Bernabe, Currier et al. 2002) (van Reeuwijk, Janssen et al. 2005), or POMGnT1 (Biancheri, Bertini et al. 2006) cause various types of MD, respectively. 4) The defects of other proteins involved in the ECM-cytoskeleton linkage such as α7 Integrin (Mayer, Saher et al. 1997) and Plectin (Smith, Eady et al. 1996) result in MD associated with delayed motor milestones and skin blistering, respectively.
Dystroglycanopathies
Hypoglycosylation of α-DG results in a group of muscular dystrophies with a wide spectrum of clinical manifestations from mild form of Limb-girdle muscular dystrophy 2I to Walker-Warburg syndrome that affects the development of muscles, brain, and eyes and results in death before age 3. There are 15 types of MD are due to hypoglycosylation of α-DG so called dystroglycanopathies, which are caused by the genetic defects of genes involved in the biosynthesis of the FOG of α-DG namely: FKTN/Fukutin (Kobayashi, Nakahori et al. 1998), FKRP (Esapa, Benson et al. 2002), LARGE (van Reeuwijk, Grewal et al. 2007), POMT1/2 (Beltran-Valero de Bernabe, Currier et al. 2002) (van Reeuwijk, Janssen et al. 2005), POMGnT1 (Biancheri, Bertini et al. 2006) POMTGnT2/GTDC2 (Manzini, Tambunan et al. 2012), B3GNT1 (Buysse, Riemersma et al. 2013), TMEM5 (Vuillaumier-Barrot, Bouchet-Seraphin et al. 2012), GMPPB (Carss, Stevens et al. 2013), PMOK/SKG196 (Yoshida-Moriguchi, Willer et al. 2013), DPM1, 2, 3, (Lefeber, Schonberger et al. 2009; Barone, Aiello et al. 2012; Yang, Ng et al. 2013) and ISPD (Ackroyd, Skordis et al. 2009). It is anticipated that more types of dystroglycanopathies will be identified in the future since a large number of genes have been identified involved in the biosynthesis of the FOG on α-DG (Jae, Raaben et al. 2013)
Duchenne and Becker Muscular Dystrophies
The Duchenne and Becker muscular dystrophies or DMD and BMD are caused by the genetic defects at the sante gene, DMD. DMD is the most common genetic disease since DMD is the largest gene in the human genome and account for about 1% of human genome, which is located on X chromosome. Thus both of the MDs occur almost exclusively in males. Both MDs have similar signs and symptoms but differ in their severity, age of onset, and rate of progression. DMD boys show muscle weakness in early childhood and worsen rapidly. They are usually wheelchair-dependent by adolescence. The BMD boys usually have milder and more varied symptoms. In most cases, muscle weakness becomes apparent later in childhood or in adolescence and worsens at a much slower rate. Both MDs are associated with a heart condition called cardiomyopathy which typically begins in adolescence. Later, the heart muscle malfunction becomes life-threatening in many cases. The DMD boys typically live into their twenties, while BMD boys can survive into their forties or beyond. The prevalence of DMD/BMD is about 1:3500 and 1:100,000 of birth of boys, respectively.
Metastatic Cancers
According to the world health organization report, estimated 14.1 million new cancer cases and 8.2 million cancer-related deaths occurred in 2012, compared with 12.7 million and 7.6 million, respectively, in 2008. It is estimated that in 2012, there were 32.6 million people (over the age of 15 years) alive who had had a cancer diagnosed in the previous five years. It is projected that 19.3 million new cancer cases per year are expected by 2025, which is due to growth and aging of the global population. According to the cancer facts & figures 2014 by the center of disease control (CDC) of USA, about 1,665,540 new cancer cases are expected to be diagnosed in USA and about 585,720 Americans are expected to die of cancer, almost 1,600 people per day in 2014. Obviously, there is an urgent need for effective therapeutic solutions to combat the cancers.
Most cancers (>90%) are derived from epithelial cells and the cancer related death are largely associated with cancer metastasis. Several genes have been identified to be associated with epithelial derived cancer metastasis including Cadherins and Dystroglycan. Increasing evidence suggested that various type of cancers found missing or dramatically reducing the FOG of α-DG on the cancer cell surfaces (Sgambato, Migaldi et al. 2003) (Sgambato, Camerini et al. 2007; Sgambato, De Paola et al. 2007). The FOG of α-DG expression level is reversely correlated with malignancy and progression of various types of cancer (Dobson, Hempel et al. 2012). The lack of the FOG of α-DG on cancer cells is due to silence of glycosyltransferases gene involved in the biosynthesis of the FOG of α-DG, such as LARGE in the cancer cells. But the detailed mechanism by which cells silence the gene expression remains unknown (de Bernabe, Inamori et al. 2009). However, restoration of the FOG of α-DG by introducing the corresponding silenced genes in the cancer cells can inhibit cancer cells growth and metastasis in mouse models (Bao, Kobayashi et al. 2009). Thus restoring the FOG of α-DG on cancer cells becomes a potential approach to treat metastatic cancers associated with loss of the FOG on the cell surfaces.
Therapy for Muscular Dystrophies
Up-to-date, there is no effective treatment for any types of MD except palliative therapy. The current efforts in drug discovery for DMD mainly based on exon skipping. The results obtained by several clinic trials are not effective and still in debate. In addition, exon skipping drug are oligo nucleotides based, which can only treats a group of specific mutations with limited scope of patients (<10% DMD). It has been reported that increasing glycosylation of α-DG can inhibit the dystrophic phenotypes in Mdx, dyW, or Sgca-/- MD mouse models by overexpressing a glycosyltransferase, Galgt2 in the mice (Nguyen, Jayasinha et al. 2002; Xu, Chandrasekharan et al. 2007; Xu, DeVries et al. 2009). As a means of increasing glycosylation of α-DG, U.S. Pat. No. 8,119,766, proposes introducing the LARGE (or LARGE2) gene to muscles, which are glycosyltransferases involved in the biosynthesis of the FOG of α-DG. However, such methods are limited by lack of effective means of delivering genes to human body. The promise of gene therapy has yet to be fulfilled due to multiple hurdles including poor uptake of the delivery vehicles and human immunological response to the gene therapeutic agents. Furthermore, the synthesis of the FOG of α-DG is a complicated process with more than a dozen genes direct involvement. The balance of the expression levels of the genes is critical for the concert biosynthesis. Simply overexpressing one of these genes may even has adverse impacts on the biosynthesis of the FOG of α-DG. In fact, independent reports suggested that overexpressing of the Large alone exacerbate the dystrophic phenotypes rather than benefit the MD mice with FKRP deficiency (Saito, Kanagawa et al. 2014) (Whitmore, Fernandez-Fuente et al. 2014). Thus new strategy to treat these diseases is much needed. Our strategy is to enhance the FOG of α-DG to strength the linkage between ECM to cytoskeleton and sarcolemma to treat various types of MD.
In mammalian, there are several linker proteins bridges DAGC to cytoskeleton such as dystrophin, utrophin, and plectins. In mdx mice, utrophin is up-regulated, which compensates the lack of dystrophin. Thus mdx mice have much milder dystrophic phenotypes compared to the human DMD patients and a normal life span, while in human there is no up-regulated utrophin in skeleton muscle. Double null of dystrophin and utrophin in mice closely resemble the clinic manifestations in human. Moreover, overexpressing of utrophin can inhibit dystrophic phenotype in mdx mice. These results suggested that alternative linker protein can compensate the defective linkage to a certain extent. Thus enhancing the connection between ECM to cytoskeleton in DMD with alternative linker protein is a promising avenue to treat DMD. However, it has been prove to be very difficult to do so. We discovered that enhancing the FOG of α-DG results in increasing the recruitment of a number of DAGC associated such as linker proteins dystrobrevin and plectin-1 to DAGC complex in muscle cells. It is conceivable that this recruitment would strength the linkage between ECM to the cytoskeleton and inhibit dystrophic phenotypes since DAGC play important roles in linking ECM to cytoskeletons and muscle function. Defects of this linkage is in common of various MDs, which has been illustrated that mutation of Dystrophin and Plectin-1 results in muscular dystrophies (Hoffman, Brown et al. 1987) (Smith, Eady et al. 1996; Rezniczek, Konieczny et al. 2007). Thus enhancing of FOG of α-DG may be an avenue to treat various MDs.